Coronaviruses are a family of enveloped RNA viruses that cause respiratory, enteric, and neurologic diseases in mammalian and avian hosts. In humans, three coronaviruses are responsible for upper respiratory tract infections; a fourth human coronavirus is the recently discovered causative agent of SARS. The genomes of coronaviruses are the largest among all the RNA viruses, which has made their genetic manipulation a formidable problem. Our laboratory developed the earliest reverse genetic system for coronaviruses, called targeted RNA recombination, with the prototype coronavirus mouse hepatitis virus (MHV). This method exploits the high rate of RNA recombination in MHV to transduce site-specific mutations into the viral genome by recombination with synthetic RNA introduced into infected cells. Coupled with a powerful host-range-based selection system, targeted RNA recombination has become a robust and versatile technique that has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the complex mechanism of coronavirus RNA synthesis. The major object of this proposal is to further extend our genetic studies of coronavirus assembly mechanisms. Targeted RNA recombination and complementary biochemical analyses will be used to answer basic questions about the functional roles of MHV structural proteins in viral replication: how the small envelope protein cooperates with the membrane protein to drive formation and budding of the virus envelope; how the spike glycoprotein endodomain specifies its inclusion in virion assembly; and how the nucleocapsid protein binds to genomic RNA and is selected for incorporation into virions. An understanding of the molecular biology of coronaviruses is critical for their control and prophylaxis. The proposed studies will provide fundamental insights into the coronavirus life cycle, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.